Intracellular delivery of biologically significant species is a vital technique to study intracellular processes and biophysical properties of living cells (Stephens, D. J. & Pepperkok, R. Proc. Natl. Acad. Sci. U.S.A. 98, 4295-4298 (2001)). Previous intracellular delivery methods include carrier-mediated methods, such as viral vectors and cationic lipids and polymers, and physical methods, such as microinjection, electroporation, and laser irradiation.
The carrier-mediated methods are effective in delivering molecules into an entire cell population, but they often associate with undesired side effects (e.g., viral toxicity, mutagenicity, and host-immune responses), endocytic degradation and trapping, and cell-type dependent efficiency, partly because of their use of active transfer mechanisms of cells (Luo, D. & Saltzman, W. M. Nat. Biotechnol. 18, 33-37 (2000), Ruan, G., Agrawal, A., Marcus, A. I. & Nie, J. Am. Chem. Soc. 129, 14759-14766 (2007)).
Several physical techniques have been proposed which involve mechanical disruption of the membrane. For example, a cell may be loaded through a microinjection technique in which the cell membrane is penetrated with a needle and macromolecules are injected into the cytoplasm through the needle. Cells may also be loaded through wounding of cells with glass beads (McNeil and Warder, 1987, J. Cell Science, 88, 669-678, through scraping adherent cells from a substrate (“scrape loading”, McNeil et al., 1984, J. Cell Biol., 89, 1556-1564), or by “pricking” cells with a needle (Yamamoto et al., 1982, Exp. Cell Res. 142, 79). Physical methods such as microinjection and laser irradiation can circumvent the cell-type dependency, but generally aim to deliver molecules into targeted individual cells. More recently, nanotechnology-based tools, such as atomic force microscopy and nanoneedles, have also been used to intracellular delivery. These new delivery methods use nanoscale tools (e.g., nanotube-based nanoneedles) that can be operated with nanoscale resolutions and thus allow high precision delivery, enabling new experimental strategies that require to manipulate living cells with high spatial and temporal resolutions and with minimal invasiveness. For example, nanoneedle-based delivery of quantum dots into the cytoplasm and nucleus of living cells has been demonstrates, made possible due to the high-aspect ratio nanoneedles with nanoscale diameters (<˜100 nm) (4-6) (Derfus, A. M., Chan, W. C. W. & Bhatia, S. N., Adv. Mater. 16, 961-966 (2004), Knoblauch, M., et al. Nat. Biotechnol. 17, 906-909 (1999), Tirlapur, U. K. & Konig, Nature 418, 290-291 (2002)).